Age-related changes in circadian regulation of the human plasma lipidome

Aging alters the amplitude and phase of centrally regulated circadian rhythms. Here we evaluate whether peripheral circadian rhythmicity in the plasma lipidome is altered by aging through retrospective lipidomics analysis on plasma samples collected in 24 healthy individuals (9 females; mean ± SD age: 40.9 ± 18.2 years) including 12 younger (4 females, 23.5 ± 3.9 years) and 12 middle-aged older, (5 females, 58.3 ± 4.2 years) individuals every 3 h throughout a 27-h constant routine (CR) protocol, which allows separating evoked changes from endogenously generated oscillations in physiology. Cosinor regression shows circadian rhythmicity in 25% of lipids in both groups. On average, the older group has a ~14% lower amplitude and a ~2.1 h earlier acrophase of the lipid circadian rhythms (both, p ≤ 0.001). Additionally, more rhythmic circadian lipids have a significant linear component in addition to the sinusoidal across the 27-h CR in the older group (44/56) compared to the younger group (18/58, p < 0.0001). Results from individual-level data are consistent with group-average results. Results indicate that prevalence of endogenous circadian rhythms of the human plasma lipidome is preserved with healthy aging into middle-age, but significant changes in rhythmicity include a reduction in amplitude, earlier acrophase, and an altered temporal relationship between central and lipid rhythms.

I would like to compliment the authors on their comprehensive work and the way they were able to describe the data in a constructive manner. I have no further comments and therefore would like to endorse this manuscript for publication.
Reviewer #2 (Remarks to the Author): There is a growing recognition of the link between circadian rhythm disruption and metabolic dysregulation and disease. This is an increasing concern within our society, particularly for the elderly. Therefore, studies exploring this link are of importance. In this study, Rahman and colleagues investigated the effects of healthy ageing on the circadian rhythms of human plasma lipidome. Their results support the main conclusion of their study, indicating that intrinsic circadian rhythm in the human plasma lipidome is preserved during healthy ageing, but its characteristics (amplitude and phase) are significantly altered.
The experimental design and analyses are appropriate, and, in most places, the results are wellreported. Overall, the manuscript is well and succinctly written and the message is relevant and timely. However, there are a few avenues that I would like the authors to consider for improvement.
1) The number of male and female participants -for completeness the authors should consider categorically stating the number of males and females used. For example, on Page 6: the sentence beginning "The goal was addressed……..(12 individuals per group, 4 and 5 females in the younger and older age groups, respectively)………". The numerical calculation for the participants here is difficult to follow when first read. This also extends to the abstract and some sections in the methods. 2) Why were the ambient lighting conditions different for young and older cohorts during the CR? Would this not confound the comparisons? 3) Related to the above, the authors should consider stating clearly in the methods the LD cycle or constant conditions that the participants experienced at specific stages in the study. 4) In the discussion, the authors focused on central clock mechanisms to explain some of the altered circadian rhythm characteristics in the plasma lipidome. Is there any study on the ageing peripheral clock activity that could also explain some of the disruptions seen? 5) Related to the above, I find the discussion rather long, and perhaps the authors should consider streamlining it. 6) Would it make sense to combine the Cosinor plots in Figures 2 and 3 to produce a single figure (in different colours)? Especially for panels A, B, and C (combining D may make the figure too busy). Minor 7) Figures: Some of the shades used to discriminate between groups are hard to see in the figures (for example figure 1). Could these be plotted in colour? 8) In figure 2 legend "The 95% CI region of the regression…." define "Cl" (which I assume is the confidence interval). Also, would be helpful for the reader if the authors write the lipid names in full at the end of this legend. 9) Brief legends for tables will be very helpful, especially if the full definitions of acronyms used are included. 10) Typological errors: For example, Page 27: -last paragraph 3rd line. 11) A few punctuation errors, such as on page 20, second paragraph, line 12.

Reviewer #3 (Remarks to the Author):
This is a very elegant and timely study characterizing peripheral circadian rhythmicity in the plasma lipidome as a marker of systemic metabolism, which potentially may be altered with aging. The strength of the article, in my opinion, is that the assessment is of healthy aging. Lipidomics analysis was performed on plasma samples 'collected in 12 healthy young [hereafter "younger", 4 females, mean±SD age: 23.5±3.9 years] and 12 healthy middle-aged [hereafter "older", 5 females, 58.3±4.2 years] individuals every 3 h throughout a 27-h constant routine (CR) protocol, which allows separation of behaviorally-induced changes in physiologic outcomes from endogenously generated oscillations in them'. The only concerns are : low n number may contribute to low power for the statistics. The Analysis only includes measurements of melatonin, which seems very odd. As metabolism is objectively regulated by cortisol, and cortisol circadian rhythm is described as an important factor in the introduction of the paper, I find it very unusual that there are no Cortisol measurement of the subjects in the study. In particular, during the CR protocol, it will be important to know whether the different cohorts respond differently to the CR regime. For example, if the 'Older' group have a different cortisol response to the CR protocol that the 'younger' group' then this may affect their metabolic profiles. If blood samples (or even saliva samples) are available, then cortisol measurements should definitely be included in the analysis, and will hopefully aid in the interpretation of any differences between 'young' and 'older' groups Minor comments: There were a many typographical errors in the manuscript. However without line numbering, it becomes a somewhat arduous task to give feedback on this. I am happy to do so in the next draft if line numbers are included. Second minor comment : Titles and legends of figures should be more informative. Many abbreviations are used (and rightly so) but a reference point (even in a supplementary figure) might help readers in 'following' and interpretation of this data-rich article.

2) Why were the ambient lighting conditions different for young and older cohorts during the CR? Would this not confound the comparisons?
Response: The lighting conditions were different between the studies as they were designed to be consistent with other studies that each were run in parallel with. Although the ambient light levels were higher in the older cohort [0.0087 W/m 2 (~3.3 lux)] than in the younger cohort [approximately 0.001 W/m 2 (~0.5 lux)], light levels for both the younger and older groups were significantly lower than the threshold of inducing circadian changes including circadian entrainment in humans and can be considered biologically inert (Gronfier et al., PNAS 2007;Zeitzer et al., 2004); therefore, we do not expect any confounding from the marginally different lighting levels. We have added the following section to the discussion to address this issue (Page 23, Lines 9-15): "Furthermore, there were marginal differences in ambient conditions between the two studies; for example, light levels were higher in the older cohort [0.0087 W/m2 (~3.3 lux)] than in the younger cohort [approximately 0.001 W/m2 (~0.5 lux)]. Both these levels, however, were significantly lower than the threshold of inducing circadian changes including circadian entrainment in humans and can be considered biologically inert (Gronfier et al., PNAS 2007;Zeitzer et al., J Physiol 2001 ); therefore, no confounding is expected from these marginal differences."

Response:
We have added light/dark (LD) cycle information to the protocol rasters that were presented in Supplemental Figure

4)
In the discussion, the authors focused on central clock mechanisms to explain some of the altered circadian rhythm characteristics in the plasma lipidome. Is there any study on the ageing peripheral clock activity that could also explain some of the disruptions seen?
Response: To our knowledge, this is the first demonstration of aging-related changes in endogenously regulated circadian peripheral rhythms in humans. A recent report (Talamanca et al., Science 2023) examining 24-hour rhythmicity in gene expression profiles from RNA-seq samples collected from 914 donors across 46 tissues found that rhythmicity was largely conserved across tissues in both younger (38 ± 9 years) and older (65 ± 3 years) groups, rhythmic gene expression was largely damped in older individuals. Importantly, however, these rhythms were not assessed under constant routine conditions, which precludes conclusively determining whether these observed changes are strictly due to changes in endogenous circadian regulation with aging or the reflect changes in the impact of external behavioral and environmental influencers (e.g., sleep/wake, activity, feeding) on circadian regulation and peripheral clocks in humans. Nonetheless, these results are largely consistent with our findings that endogenous circadian regulation of peripheral rhythms in lipids is conserved across aging but significantly damped (i.e., lower amplitude) in older humans. Furthermore, both of our results are consistent with previous reports from studies in animal models that show that aging affects peripheral clocks largely with phase advances and damped oscillations, which, data suggests, is due to reduced coupling strength of the SCN to peripheral tissues (Yamazaki et al., PNAS 2002;Tahara et al., Aging 2017). We have included this discussion in the revised report (Page 19, Lines 9-24).

5) Related to the above, I find the discussion rather long, and perhaps the authors should consider streamlining it.
Response: We have revised the discussion section throughout to consolidate and reduce the overall length.
6) Would it make sense to combine the Cosinor plots in Figures 2 and 3 to produce a single figure (in different colours)? Especially for panels A, B, and C (combining D may make the figure too busy).

Response:
We have edited both Figure 2 and 3 to move the exemplary cosinor plots into a combined new Supplemental Figure 2. We have chosen to keep the panels for the younger and older groups separate as overlaying the plots even as different colors makes it difficult to discern the individual plots and corresponding 95% CI bands.
Revised Supplemental Figure figure 1). Could these be plotted in colour?

Response:
We have used color to primarily differentiate between lipid species or time points. We hope that the higher resolution images in the final version, if accepted, will help with discriminating groups plotted in black and grayscale. figure 2 legend "The 95% CI region of the regression…." define "Cl" (which I assume is the confidence interval). Also, would be helpful for the reader if the authors write the lipid names in full at the end of this legend.